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c 

P^ C H ARS 34-24 

S EA ^g~**m 

«W SERIAL RECORDS 



THE USE OF COLCHICINE IN PLANT BREEDING 



August 1961 



U S 



Agricultural 
DEPARTMENT 



Research Service 
OF AGRICULTURE 



ARS- 34-24 
August 19 bl 



THE USE OF COLCHICINE IN PLANT BREEDING 
By Haig Dermen and S. L. Emsweller— ' 

Colchicine, a poisonous medicinal chemical, has been used since 1937 in 
plant breeding work to produce changes in plants by doubling the number 
of chromosomes in cells, a condition referred to as polyploidy. The 
increased number of chromosomes usually brings about an increase in size 
of the affected cells and various degrees of changes in their functions. 
In contrast with the normal plants, those developed by colchicine treatment 
often show changes in height and width; in thickness of branches; in size, 
shape, and texture of leaves, flowers, fruits, and seeds; in fertility of 
flowers; and in physiological responses, However, the degree of changes 
produced when the chromosome number is doubled cannot be predicted, and 
"magic" changes referred to by some popular writers are not to be expected. 
The visual changes induced in some plants are so small that even an 
experienced person will have difficulty in recognizing them. 

Colchicine is extracted from either the seeds or the corms of Colchicum 
autummale L. (meadow saffron or fall crocus) and may be bought in very 
small quantities in powder form from certain chemical concerns mentioned 
at the end of this article. A small quantity of colchicine can be used 
to treat a large number of plants, seeds, or other types of material, if 
applied without undue waste. Where a dipping method is used, the solution 
may be used several times. Generally colchicine is used in water solution 
in concentrations from as low as 0.1 percent up to 1.00 percent; it also 
can be used in 10-percent glycerine in water solution. The assistance of 
a druggist may be obtained in preparing desired percentages and quantities 
of solutions. 

In handling colchicine, extreme care should be taken to-- 

Keep it out of the eyes to avoid possible dangerous consequence. 
Wash hands after contact with the chemical to prevent possible 
skin irritation. 

HOW COLCHICINE WORKS 

The colchicine solution affects plant cells during the division stages and 
has no apparent effect on nondividing cells. In normal cell divisions the 
chromosomes split lengthwise, and each half chromosome migrates to the 
opposite sides of the cell. A new cell wall forms between the two masses 
of chromosomes and thus two daughter cells are formed, each having the 
same number of chromosomes as the mother cell. If colchicine is 



1_/ Cytologist and principal horticulturist, Crops Research Division, 
Agricultural Research Service, U. S, Department of Agriculture, 
Beltsville, Md. 



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absorbed into the dividing cell, the chromosomes split but the split 
halves do not migrate to the opposite sides, and the division of the 
cell is prevented. Thus, from the colchicine treatment there results 
a cell with twice as many chromosomes in its nucleus as the same cell 
had previous to treatment. After such initial chromosome doubling, if 
the colchicine diffuses away from the plant tissues or becomes so dilute 
as to be ineffective, the nuclei in the cells with the double chromosome 
number can divide normally to form daughter cells but with the double 
number of chromosomes. In addition to doubling the number of chromosomes, 
colchicine treatment has at times been found to cause addition or loss of 
one or more chromosomes in both the normal and the doubled types. Some 
abnormalities appearing on some treated plants may be caused by it. It 
is suspected that colchicine may also cause gene mutation, but there is 
considerable doubt about this. It is obvious from the way colchicine 
affects plant cells that applications will be effective if the material 
to be treated is growing vigorously, so that many cells are dividing. 
Treatment of dormant seeds, scales of lily bulbs, and similar material, 
however, has induced polyploidy, presumably because colchicine penetrates 
the seeds and scales and is maintained there for some time until cell 
divisions have begun; after this the colchicine becomes effective. 

TYPES O F CHANGES INDUCED 

In the terminal growing points of shoots and in lateral buds, there appear 
to be three cell layers each of which gives rise to a separate tissue or 
a tissue system or to areas in a tissue in the stem, leaves, flowers and 
fruits. The outer cell layer, 1st layer (L-I) of the growing point of 
the shoot or bud generally gives rise to a one-cell thick tissue, the 
epidermis. The next layer, 2nd layer (L-II) may give rise, depending 
on the kind of plant, to either a layer of tissue one-cell thick immediately 
under the epidermis, or to several layers of the cortical tissue. A third 
layer (L-III) may give rise to the remaining tissues in the stem. Pollen 
grains and egg cells originate in the tissue of anthers and ovules, 
respectively, derived from L-II. For a fuller discussion of this subject, 
the reader is referred to articles 5 and 8 in the list of the literature 
given at the end of this article. 

Colchicine treatment may affect one cell layer and not the others, or any 
two layers may be affected and the remaining layer not be affected, or all 
three may be affected. This last possibility is rarer than the first two. 
Furthermore, many of these layers may have become only sectorially polyploid. 
Experiments have shown that polyploidy resulting from colchicine treatment 
may be of three main types: 

1. Epidermal -polyploidy . Enlargement of stomates (breathing pores) on 
leaves subsequent to treatment indicates that the epidermis or outermost 
layer of the material is polyploidized, but it does not indicate that 
internal tissues are changed. If the internal tissues are not polyploidized, 
this type of change may be called epidermal-polyploidy . 



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2. Internal -polyploidy . Any change in which a layer of cells or a tissue 
or tissues beneath the epidermis are made polyploid but in which the 
epidermis remains normal may be called internal-polyploidy . This type of 
polyploidy may be of three kinds: (a) only the tissue immediately next to 
the epidermis is polyploidized; (b) the inner tissues are polyploidized, 
but the tissue for a number of cell layers beneath the epidermis remains 
normal; (c) all the inner tissues become polyploidized, the epidermis 

of the plant alone remaining normal. Enlarged pollen grains from flowers 
of a treated plant indicate that at least the tissue next to the epidermis 
is polyploidized from the colchicine treatment. Leaves become larger and 
broader in any kind of internal polyploidy. Flower petals and especially 
the anthers are chiefly affected if the tissue next to the epidermis is 
polyploidized. Chromosome counts in the cells of roots of a vegetative 
cutting would show whether or not the conductive tissue of the stem is 
polyploid. Such an examination of roots of seedlings treated by colchicine 
does not indicate whether the aboveground portion of a seedling is affected 
by colchicine treatment or not. 

3. Total -polyploidy . A plant in which all the tissues are polyploidized 
may be designated as total-polyploid. 

The extent of polyploidy can best be determined by examining, under a 
high-power microscope, parraffin sections of treated material taken 
from the very tip of a vigorously growing branch suspected of polyploidy. 
In such preparations, one must determine the chromosome number in a 
number of cells in each tissue system mentioned above to find out exactly 
what tissue system is polyploidized. Size of cells or of nuclei or of 
mere chromatin mass alone is not sufficient to determine the absolute 
extent of polyploidy in the treated plant. 

From the results on induced polyploids it has been shown that the degree 
of fertility of these plants is usually affected. In a number of cases 
sterile hybrids resulting from hybridization between species of the same 
or closely related genera have been made fertile by chromosome doubling, 
while with others that has not been possible. Often where the plants 
have been highly fertile, they are made less fertile. Lessening of the 
fertility or inducing complete sterility by polyploidy may not always be 
deleterious. Where, for instance, plants are propagated vegetatively and 
grown for leaves and flowers or other plant parts, sterility may even be 
desirable. In such fruits as strawberries and raspberries, lowering of 
fertility causes numerous seeds to fail to develop and results in partly 
developed and deformed fruits, However t such plants may become of great 
value as breeding material. 

A number of reports indicate that besides quantitative changes in plants 
resulting from polyploidy there also have been changes of qualitative 
character. These may be in color intensity of leaves and flowers; in 
fragrance of leaves, flowers, or other plant parts; and in content of 
oils, starches, sugars, and vitamins. Fundamentally these changes that 
are ordinarily considered qualitative are mostly, if not entirely, changes 
of quantitative nature, since they result from either an increase or a 
decrease of the various products naturally present in the plants. 



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METHODS OF TREATMENT AND DETECTION OF POLYPLOIDY 

The colchicine solution at the beginning and during the tine of treatment 
should be at room temperature. With small seedlings or fast-growing 
herbaceous plants it is best that the treatment be made with as weak a 
solution as may be effective, as early in the active growth of the plant 
as possible, and for a relatively short period of time. Application of 
a strong solution or a prolonged treatment may prevent growth of the 
treated material and kill it. Success in the treatment of growing material 
is based on the principle that the chemical should reach the regions of 
growth; therefore, the xaanner of treatment depends upon the type of 
material to be treated. A few of the methods, which may be modified to 
suit the material and the inclination of each experimenter, follow: 

1. Seed treatment . Seeds of many varieties of plants have been soaked 
in a 0.2 percent to 1.6 percent (usually 0.2 percent is sufficient) 
solution for 1 to 10 days before planting. Soaking of seeds should be 
done in a very shallow container so that the seeds are not deprived of 
oxygen. Seed treatment methods should be used only for seeds that germinate 
quickly. In the case of slow-germinating seeds, the treatment should be 
deferred until the seeds commence active germination, in which case the 
treatment is fundamentally the same as with seedlings. 

2. Seedling treatment . Freshly germinated seeds are kept immersed in 
about 0.2 percent colchicine solution in a shallow container, or placed 
on filter paper or blotting paper spread out in shallow dishes and 
thoroughly wet with the solution, for 3 to 24 hours or longer, depending 
upon how rapidly the seedlings are growing and how bulky they are. If 
the material is suitable, the young seedlings may be treated as follows: 
The root ends placed on a strip of absorbent cotton that is thoroughly 
wet with water and then the seedlings rolled into a bundle. The cotton 
covers the root ends and forms a plug that will fit loosely in a small 
vial. The bundle of plants is then inverted and set in the vial with 
only the stem ends immersed in the water solution of colchicine. By 
this method the root system is kept moist only with water and practically 
unaffected by the chemical, thus preventing some of the mortality that 
usually follows treatment of whole seedlings. When seeds are germinated 
in the dark the resulting seedlings, being longer than normal, are often 
suitable for this type of treatment. 

Another method is: Plant seeds, well spaced, in pots or flats. When 
seedlings have emerged from the soil and the cotyledons or the first one 
or two leaves have opened, place a small amount of 0,2 to 0.5 percent 
colchicine solution between the cotyledons or over the vegetative growing 
point. To each 10 cc. of colchicine solution in water or 10 percent 
glycerine-water solution should be added 1 to 2 drops of 10 percent 
solution of Santomerse-SX (a product of the Monsanto Chemical Company, 
St. Louis, Missouri) or a similar wetting-detergent agent, such as 



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Dreft, a household detergent, at the same concentration, used as a 
substitute for soap.iL/ Leave some seedlings untreated for comparison 
with the treated ones. The growth of the treated seedlings will be 
retarded in comparison with the untreated ones if the treatment is at 
all effective. 

3. Treatment of growing shoots and buds . Tips of rapidly growing shoots, 
or an entire growing bud, may be treated by placing a drop or more of the 
colchicine solution over the tip, once or several times, or by immersing 
the growing tip for a number of hours in a vessel containing the solution. 
The tips of growing shoots may be smeared with a mixture of 0.5 to 1.0 
percent colchicine in lanolin. All these methods have given satisfactory 
results, particularly with herbaceous plants. Woody and semiwoody plants 
(buds or young shoot tips) require a somewhat different treatment. For 
these, one of the following solutions is recommended: (1) 0.5 to 1 percent 
colchicine in a 10 percent water solution of glycerine; or (2) 0.5 to 1 
percent water solution of colchicine. In order to facilitate penetration 
and spreading of these solutions, a very small amount of a wetting or 
spreading agent (see above) should be added. 

Another method is: Cut off the very tip of a vigorously growing shoot 
and wet with the colchicine solution the upper three lateral buds, 
three to eight times at 1 or 2 day intervals. Other buds either should 
be dug out or shoots growing from untreated buds should be destroyed. 
Colchicine effect should be looked for in those buds or shoot tips that 
are partly retarded in growth as a result of the treatment. The effective- 
ness of the treatment may be judged from the distorted growth of first 
few leaves emerging after the treatment. If no such distortion is 
observed in the lower leaves of the growing shoot, it is most obvious 
that colchicine solution has not penetrated into the treated buds at all. 

It is advisable to limit the treatment to buds of one young shoot on a 
cutting or grafted shoot; the rest should be cut off. Shoots that are to 
be treated should be allowed to grow some 6 inches or more before the 
tip or lateral buds are treated. The older leaves on such shoots should 
be left to furnish nutrition for the plant. 

In woody material, if not in herbaceous plants as well, total polyploidy 
affecting the whole new growth very seldom occurs. The effect often may 
be confined to a narrow sector on a branch, or to a certain tissue, or to 
a limited portion of tissues. When shoot growth from treated buds does not 
show any retardation as compared with growth from nontreated material, 
it may indicate that the treatment has not been effective. Basilary 
leaves of a shoot grown from a treated bud appear distorted in varying 
degrees, when the treatment has been effective. 



2/ Mention of companies or products is not to be construed as an 
endorsement of these firms or products by the U. S. Department 
of Agriculture. 



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Colchicine-induced polyploidy can be often recognized in leaves that are 
denser in texture, darker in color, larger and broader than normal, or 
in part of a leaf (often a half, one side of the midrib) that is larger 
than the other part and denser in texture. Such leaves should be looked 
for in the upper portion of the shoots that develop from the main growing 
point of the treated tips or from axillary buds that were present in the 
treated regions. Polyploid branches may be grown from buds in the 
axils of the changed leaves by cutting off the part above these leaves. 
If no change is detected, then buds that were very young and were 
developing at the time of treatment and which may subsequently have 
remained dormant may be forced into growth on a possible chance that 
they have been affected by the treatment and contain polyploid changes. 
Any extraneous growths that may hinder growth in the treated part should 
be constantly eliminated. 

When a polyploid sector is discovered in an otherwise normal stem, it 
should, if possible, be forced to grow out into a branch; the propagation 
of such a branch by grafting or rooting (depending on the type of plant) 
should not be delayed. A polyploidized sector as such may not continue 
to grow indefinitely parallel with a normal tissue in the same stem, and 
eventually may be lost as the stem develops. 

4. Treatment of bulbs and corms . The terminal growing points in bulbs 
and corms are buried within a mass of scales or other storage tissues and 
are, therefore, difficult to reach with colchicine. In the case of tulips, 
narcissus, bulbous iris, and onion the colchicine may be introduced into 
the growing regions by a hypodermic needle. In gladiolus, penetration 

of colchicine solution into small cormels has been achieved under a 
partial vacuum. This method might prove effective also with small bulbs 
of tulip and narcissus. The "eyes" on the gladiolus bulbs may be treated 
directly, in the same way buds are treated. In bulbaceous material such 
as lilies, where detached scales from the bulb may readily form small 
bulbs at the base after planting, the following treatment has been very 
successful: Scales are detached and immediately immersed in a 0.2 percent 
solution for about 2 hours; then they are planted with the tips exposed. 
The length of time of treatment and concentration of the solution may 
have to be varied to obtain results with the different species of lilies 
and other bulbaceous material. 

5. Treatment of day-lilies and similar types of plant s. Many herbaceous 
plants form large root stocks or crown from which new buds arise each 
year. These new buds originate from cells within but near the surface 

of the crown tissue, and if colchicine is present at the time bud differ- 
entiation is initiated, some of the buds may be expected to give rise to 
polyploid shoots. The daylily is such a plant and may be treated by 
cutting the tissue back deep into the crown and scooping out a shallow 
basin in x^hich the colchicine solution can be placed. It has been found 
that colchicine in concentrations of 0.1 to 0.5 percent have 



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been effective. The colchicine has been used in water solutions and 
in lanolin. It should also be effective in 10 percent glycerine. 
Colchicine-lanolin paste is applied only once. It is advisable to 
repeat the treatment with colchicine solution in water or glycerine 
every few days for three to four treatments to be sure enough of 
the drug enters the crown of the plant. As the new buds develop, 
some will be affected as evidenced by their slow growth as compared 
to unaffected buds. The latter should be removed as soon as possible 
to force the polyploid bud into growth. 

If polyploid shoots are obtained, it must be remembered that they are 
on the old diploid root system. Such shoots may form small side shoots 
at the nodes and these should be removed and rooted in order to obtain 
a plant with a polyploid root also. 

At this point it must be emphasized that desirable changes in many plants 
are not to be expected, since some of them are already polyploids and 
further doubling of the chromosomes may result in inferior plants. Also, 
it cannot be emphasized too strongly that colchicine treatment frequently 
has been unsuccessful, especially with woody plants and other material 
where penetration of the chemical into the growing regions presents 
difficulties; and furthermore, that the doubled-chromosome forms, if 
produced, are not always improvements over the normal types. Some 
changes often can be recognized only by careful observation, involving 
the use of a microscope or a high-magnifying hand lens. Some changes 
effected by colchicine may be so inconspicuous that they at times 
escape casual observation. In interpreting results from the use of 
colchicine it is not only necessary to know the growth habits and 
cultural requirements of the experimental plant but also to have 
expert knowledge of plant structure, some knowledge of the principles 
of genetics, experience in recognizing and evaluating induced changes, 
and training in the use of the microscope. A high-magnifying hand lens 
in some cases may make it possible to see a change in size and distribution 
of stomates in comparison with stomates of a normal leaf. Changes in 
superficial structures such as hairs and glands that are of epidermal, 
origin may also be detected. No trace of colchicine is expected to be 
present in parts of polyploid plants resulting from the experiments. 

As can be seen from the above presentation, colchicine cannot be con- 
sidered as a plant food, "growth elexir," or fertilizer. Its application 
to induce polyploidy is confined mostly to vegetative growing points of 
individual plants, and only a very limited number of plants is treated 
at a time. For a more extended discussion of artificially induced 
polyploidy and information on some of the results obtained, readers 
are referred to the limited list of literature references at the end of 
this paper, particularly to reference 4, "Colchicine Polyploidy and 
Technique", which is essentially a general review of the literature on 
the subject. The publications listed may be found in the libraries of 
most State agricultural colleges and experiment stations. 



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NOTE: Colchicine may be purchased from following chemical concerns:—' 
Inland Alkaloid Company, Tipton, Indiana; Mallinckrodt Chemical Works, 
New York City; Eimer & Amend, New York City; Merck & Co., Inc., Rahway , 
New Jersey; S. B. Penick & Company, 132 Nassau Street, New York City; 
Gane and Ingram, Inc., 43 W. 16th Street, New York City; Bios Laboratories, 
607 West 43rd Street, New York 18, New York 



3/ On furnishing the names of manufacturers of colchicine, it is impracticable 
to provide a complete list. These references are given for the convenience 
of the reader, with the understanding that no discrimination is intended 
and no guarantee implied by the U. S. Department of Agriculture. In order 
to find out who has colchicine available for sale in small quantities, 
one should write to all of the above companies, because some may have 
discontinued handling it. 



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REFERENCES 



1. 



2. 



4. 

5. 

6. 

7. 

8. 

9. 
10. 
11. 
12. 



Beasley, J. 0. 
Blakeslee, A. 

Dermen, Haig. 



Emsweller , S 



The production of polyploids in Gossypium. Jour. 
Heredity 31: 39-48, 1940. 
F. , and Avery, Amos G. Methods of inducing doubling 
of chromosomes in plants by treatment with colchicine. 
Jour. Heredity 28: 393-411, 1937. 
A cytological analysis of polyploidy induced by 
colchicine and by extremes of temperature. Jour. 
Heredity 29: 211-229, 1938. 
Colchicine polyploidy and technique. Bot. Rev. 6: 

599-635, 1940, 
Periclinal cytochimeras and histogenesis in cranberry. 
Amer. Jour. Bot. 34: 32-43, 1947. 
Colchiploidy in Grapes. Jour. Hered. 45: 159-172, 
1954. 

Sterile hybrid grape made fertile with colchicine. 

Fruit Varieties and Horticultural Diges, 12: 34-36, 1958, 
Nature of plant sports. The Amer. Hort. Mag. 39: 123- 

173, 1960. 

L. The utilization of induced polyploidy in Easter lily 
breeding. Amer. Soc. Hort. Sci. 49: 379-384, 1947. 
Flowers as you like them. Yearbook of Agriculture, Pages 

284-288, 1943-1947. 
and Brierley, Philip. Colchicine-induced tetraploidy in 

Lilium. Jour. Hered. 31: 223-230, 1940. 
and Stewart, R N, Diploid and tetraploid pollen mother 
cells in lily chimeras. Amer. Soc. Hort. Sci. 57: 414- 
418, 1951. 

Johnstone, F. E. , Jr. Chromosome doubling in potatoes induced by 

colchicine treatment, Amer. Potato Jour. 16: 288- 
304, 1939. 

Levan, Albert. The effect of colchicine on root mitosis in Allium 

Hereditas 24: 471-486, 1938. 
Colchicine-induced tetraploidy in perennial rye grass. 

Jour. Hered. 30: 499-504, 1939. 
and Ruttle, M. L. Colchicine and its place in fruit 
breeding. N. Y. State Agr. Expt. Sta. Cir. 183, 19 
pp., illus. 1938. 
The cytological and genetical significance of colchicine. 

Jour. Hered. 29: 3-9, 1938. 
Observations on the immediate effects of colchicine. 
Jour. Hered. 30: 35-37, 1939. 
Rasmussen, J., and Levan, A. Tetraploid sugar beets from colchicine 

treatments. Hereditas 25: 97-102, 1939. 
Blakeslee, A. F. , and Avery, A. G. Demonstration of the 
three germ layers in the shoot apex of Datura by means 
of induced polyploidy in periclinal chimeras. Amer. 
Jour. Bot. 27: 895-905, 1940. 



Myers, W. N. 
Nebel, B. R. 



J Mara, J. G. 



Satina, S 



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21. Satina, S. and Blakeslee, A. F. Periclinal chimeras in Datura 

stramonium in relation to development of leaf and 
flower. Amer. Jour. Bot. 28: 862-871, 1941. 

22. Sears, £. R. Amphidiploids in the Triticinae induced by colchicine. 

Jour. Hered. 30: 38-43, 1939. 

23. Smith, Harold H. The induction of polyploidy in Nicotiana species 

and species hybrids. Jour. Hered. 30: 291-306, 1939. 

24. Thompson, R. C. , and Kosar, W. F. Polyploidy in lettuce induced by 

colchicine. Amer. Soc. Hort. Sci. 36: 641-644, 1939. 



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